The invention relates to the fields of microscopy, computerized cell imaging, immunohistochemistry, histopathology, oncology, protein quantitation, and diagnosis and prognosis of cancer and other diseases.
1. Immunohistology
The presently universally-accepted method for the diagnosis of all types of solid cancer is the histologic determination of abnormal cellular morphology in surgically biopsied or resected tissue. Once removed, the tissue is preserved in a fixative, embedded in paraffin wax, cut into 5 xcexcm-thick sections, and stained with two dyes: hematoxylin for the nucleus and eosin for the cytoplasm (xe2x80x9cHandE stainingxe2x80x9d).[1, 2] This approach is simple, fast, reliable, and inexpensive.
Histopathology allows the diagnosis of a variety of tissue and cell types. By providing an estimation of tumor xe2x80x9cGradexe2x80x9d (cellular differentiation/tissue architecture) and xe2x80x9cStagexe2x80x9d (depth of organ penetration) it also makes prognosis possible.[3, 4] In the Surgical Pathology Departments of larger hospitals histologic HandE staining is generally automated, tissue-processing technique is standardized, and histologic interpretation is well established.
Aside from crude measurements of the tumor diameter, pathologists do not attempt to quantify the area or volume of dysplastic tissue, nor do they perform absolute quantification of the cancer-related proteins present in such specimens. When antibody staining is attemptedxe2x80x94commonly called immunohistochemistry (IHC)xe2x80x94the intensity and area of its visible or fluorescent color is ranked in an ordinal fashion. This ordinal ranking by the pathologist is accomplished by to the subjective impression of both the extent (area) and the darkness of the stain, compared to adjacent, morphologically normal tissue. The number of ranked categories and the cutoff points for each is arbitrary and inconsistent among observers. Also, for some organs and cancer proteins, there has been observed a xe2x80x9cfield-effectxe2x80x9d in which abnormal proteins are expressed in adjacent, supposedly negatively-stained, morphologically normal tissue.[5] Furthermore, since there is no matching of cells between the HandE histology slide and the immunostained slide, it is difficult to segregate the immuno-scoring for different histologic classes, e.g., cancer and pre-cancer, within the same section and it is impossible to accurately correlate total immunostaining with histologic area for each tissue class.
The currently available optical techniques of microscope-based cell imaging provide a partial solution to the problem of performing these cellular measurements. This approach uses conventional light microscopy combined with monochromatic light filters and computer software programs. The wavelengths of the light filters are matched to the colors of the antibody stain and the cell counterstain. The filters allow the microscopist to identify, classify and then measure differences in the optical density of specific colors of light transmitted through immunostained portions of tissue sections. See U.S. Pat. Nos. 5,235,522 and 5,252,487, both of which are incorporated herein by reference, for applications of these methods to tumor protein measurement.
More advanced cell imaging systems (image cytometers) permit automated recognition of features, and combine this with automated calculation of feature areas, automated calibration, and automatic calculation of average and integrated (xcexa3OD) optical density. (See, e.g., U.S. Pat. Nos. 5,548,661, 5,787,189, both of which are incorporated herein by reference, and references therein.) Merely scoring patient tissue immunostaining by ordinal rank, however, even by incorporating the more objective and uniform optical estimation techniques provided by Cell Imaging Densitometry (CID), provides limited information for patient and tissue evaluation. By translating such scoring into common biological units of measurement, oncologists and pathologists can refer to the patient""s particular xe2x80x9cprofilexe2x80x9d of tumor suppressor and oncogene protein levels. Thus, the clinician will be able to numerically predict a patient""s xe2x80x9crelative riskxe2x80x9d of relapse or death, probability of chromosomal instability, metastases, response to therapy, or even probable survival duration. The suggested method should also make it possible to sum up a patient""s total xe2x80x9cbody burdenxe2x80x9d of such proteins, where there are multiple lesions. A fraction of this tissue burden escapes the porous membrane of the cancer cells into the blood stream perfusing the tumor(s), achieving a steady-state concentration over time. Immunoassays e.g., ELISA, can accurately and sensitively measure these volumetric concentrations. Knowing the typical quantitative correlation between blood levels and tissue expression will allow us to more effectively (and less invasively) indirectly monitor residual/recurrent disease.
One reported attempt to improve the accuracy of the measurement of cancer protein in tissue used Western Blotting in combination with CID to create immunohistochemical rankings when measuring HER-2/c-erbxcex2-2 oncogene protein expression in breast cancer patients.[6] In this attempt, cultured human breast cancer cells were genetically-engineered in order to express different levels of the oncogene protein. HER-2 protein levels (pg/cell) in the cell lysates of these reference cells were estimated with dilutions of a fragment of recombinant purified HER-2, using the Western blot assay and laser densitometry. Cultured cell pellets were snap-frozen in xe2x80x9cOCTxe2x80x9d (polyethylene glycol-polyvinyl alchohol-trimethylbenzylammonium chloride) embedding media, cut into 4 xcexcm sections with a cryostat, and then attached to microscope slides, presumably by air drying. Breast cancer tissue was fixed in 95% ethanol, followed by buffered formalin. Alternatively, tissue from the same tumors was either frozen in OCT and cryosectioned, or paraffin-embedded and sectioned with a microtome. A CID/Western blot xe2x80x9cstandard curvexe2x80x9d on the cultured cells was created with a single immunostained CID standard, which was assumed (without testing or reference) to be 1 pg/cell. This xe2x80x9ccurvexe2x80x9d from the frozen reference cells was then applied to the immunostained breast tissue by using a single xe2x80x9ccorrection factorxe2x80x9d (xcx9c40%) in order to boost the actual optical density scores for the paraffin tissue sections. In the eventual correlation of tumor recurrence with HER-2 overexpression these xe2x80x9cquantitativexe2x80x9d immunostaining scores were, once again, reduced to ordinal ranks, xe2x80x9cLowxe2x80x9d, xe2x80x9cMediumxe2x80x9d, and xe2x80x9cHighxe2x80x9d, which reflected increasing degrees of amplification of the gene""s DNA. The authors were able to predict relative differences among the women in their risk of tumor recurrence.
However, fixation conditions of the reference cells and the tissue were different, there were no immunostained paraffin sections for the reference cells, and the frozen tissue stained more intensely than the paraffinized tissue (disproportionately so, depending upon the level of HER-2 protein overexpression). This approach provides no method to summarize the total HER-2 tumor burden per patient or tumor. The reliance upon Western blot for quantitation of the oncoprotein cell is a disadvantage, due to the complexity and slowness of the procedure, plus its modest quantitative accuracy, precision and reproducibility.[7-9] Another approach employs simultaneous measurement of nuclear DNA by cell imaging to provide an internal calibration reference (U.S. Pat. No. 5,252,487). This method is subject to variations in the intensity of the DNA staining and derives its calibration xe2x80x9ccurvesxe2x80x9d for staining intensityxc3x97pg DNA/cell from a single DNA value.
Another attempted to solve the problem was the Quicgel(trademark) method, described in U.S. Pat. No. 5,610,022, which used immobilized cultured cells as xe2x80x9cinternal controlsxe2x80x9d in order to estimate the xe2x80x9cpre-processing immunoreactivity levelxe2x80x9d of individual paraffin tissue sections.[124] The stated goal was to compensate for unpredictable and/or excessive loss of antigenicity due to fixation, which often alters the chemical structure of antigens. The internal controls were intended to provide a correction factor, allowing an estimation of the IHC staining intensity that would have been obtained with fresh, unfixed tissue. These internal controls (xe2x80x9cpseudo-tissuexe2x80x9d) were treated as though they were tissue, and were subjected to the same processing conditions experienced by a clinical tissue sample sharing the same paraffin block.
In this approach, the xe2x80x9cpseudo-tissuexe2x80x9d control cells were fixed twice. The first fixation, prior to immobilization of the cells in a matrix, was in paraformaldehyde for less than ten minutes at room temperature. This level of fixation has previously been shown to be sufficient to maintain the structure of the cultured cells while minimizing antigen diffusion and loss of immunoreactivity.[125, 126] The fixed cells were then encased in a thick, 3 mm slice of agar gel, and this xe2x80x9cpseudo-tissuexe2x80x9d underwent a second fixation in 10% neutral buffered formalin, duplicating precisely the conditions under which the tissue sample was fixed. The patent describes a test of the Quicgel(trademark) method, wherein the pseudo-tissue and tissue samples were exposed to formalin for four fixation times ranging from 4 to 72 hours, in order to determine the rate at which immunoreactivity was lost over time for both the pseudo-tissue controls and the adjacent tissue samples.
Typically, formalin fixation of patient tissue lasts 6-12 hours; the time must be adjusted for the size of the tissue specimen and the density of its tissue type (e.g., lung tissue is penetrated very quickly, while breast tissue is penetrated much more slowly).[127] The Quicgel(trademark) method attempted to correct for the resulting variations in immunostaining level by using a cell imaging densitometry program to measure overall staining area, or intensity in pixels, but did so without a standard curve. Rather, the method assumed the existence of an inverse proportionality between fixation duration and cell staining that was linear back to zero fixation time, and also assumed an equal rate of loss of immunostaining for the xe2x80x9cpseudotissuexe2x80x9d and the specimen tissue, regardless of the identity or size of the specimen. As discussed below, both assumptions are incorrect.
For these reasons, there has never been a demonstration of the Quicgel(trademark) method for correlation of protein levels with patient survival or matched blood levels, no incorporation of histologic tissue class, and no calculation of tumor burden. Others have subsequently used reference cells to standardize microscope settings and automate cell imaging scoring via gray scale tables to maximize optical density contrasts. These workers also conducted extensive fixation of the pseudotissue.[128] More recently, this same group has extended the Quicgel(trademark) approach for fixation artifact correction, testing the method with both internal controls (same paraffin block as the tissue sample) and external controls (separate paraffin block, but stained simultaneously) for the effects of fixation duration and tissue sample storage conditions. They also found fixation artifacts in cells and tissue to be subject to discontinuities.[129]
The approaches just described have deficiencies which make it impossible to standardize IHC scoring and to translate optical density pixels into absolute quantities of protein. FIGS. 19 and 20 present the data given in the description and figures of U.S. Pat. No. 5,610,022. It is apparent that the degree of loss of immunoreactivity in the pseudotissue and in the specimen tissue at different fixation times is not the same, regardless which cell imaging measurement is used. Because the loss of staining is not proportional, the pseudotissue controls cannot be used to estimate pre-processing immunoreactivity level in the breast tissue samples. This is true even within the observed range of fixation times; reliable extrapolation back to time zero is therefore not possible. This is indicated by the lack of statistical significance of the implied linear regression equations, suggested by the model used in U.S. Pat. No. 5,610,022. This is not surprising, given the relative rates of fixative penetration in the trypsinized reference cells versus the dense, stroma- and lymphocyte-laden, high-fat breast tissue samples.
To create a valid standard curve for protein quantitation, both calibration cells and specimen tissue should be subjected to the same fixative and IHC reagents, the treatments of each must be optimized with respect to such things as fixation duration and temperature, antibody concentration, and substrate incubation time. The goal is identical staining of the calibration cells and the tissue samples, not identical treatment. If the former condition can be met, the amount of target protein can be read off the cell imaging calibration cell staining curve for each IHC batch. Whether the staining of the calibration cells and the tissue is, in fact, identical, will be revealed from the similarity of their respective cell imaging:
1. signal/noise ratios;
2. frequency distributions;
3. dynamic range of protein expression; and
4. definitions of positive staining.
Fixation and staining conditions may vary with the tissue type, fixative, antibody, and IHC materials and methods used, but if identical staining between standards and specimens can be achieved, inter-laboratory results for any protein will be commensurate. The present invention, by eliminating fixation of the pseudotissue (a step heretofore assumed to be essential to the use of pseudotissue controls), provides a quantitation method that satisfies all the above criteria, and which is demonstrably superior to subjective IHC scoring in its ability to generate histologic diagnoses and target protein concentrations in blood, and to correlate them with cancer patient survival.
As recently as May 17, 1996, the American Society of Clinical Oncology summarized the current state-of-the-art in the use of tumor marker tests in prevention, screening treatment and surveillance of breast and colorectal cancers.[10] It assessed a variety of tumor markers, such as p53, CEA, and DNA flow cytometry [HER-2 was not considered]. The consensus report concluded that such markers continued to have limited prognostic or predictive value. When DNA mutations are assessed, it is often not clear which mutations have an impact upon gene function. With respect to IHC the problem is the inability to generalize among the results from different clinical trials; this is due to the variety of antibodies and lab methods used as well as the absence of a common objective criterion for xe2x80x9cabnormalxe2x80x9d staining.
There remains, therefore, a need to standardize the current method of scoring immunohistologic staining of paraffin-embedded tissue sections. This would to allow valid comparisons of results among different laboratories or among different staining batches within the same lab for any disease-related protein for which there are adequate antibodies and cultured cell lines. Such standardization would also create the conditions for direct quantitation of disease-causing antigens in patient tissue and blood. Such measurement offers the potential for determining in-situ treatment dosages as well as estimated months of patient survival. The present invention provides such a standardization method.
Although over-expression of aberrant proteins is usually multi-focal, it is also clonal in nature: abnormal proliferating cells are contiguous in their stainingxe2x80x94due to cell division from a single progenitor cellxe2x80x94and share the same proliferation behavior and a common profile of genetic defects. In the case of p53 this phenomenon distinguishes clonal expression of the mutated protein from a transitory over-expression of wild-type protein in an occasional cell in which the tumor suppressor response has been elicited. The latter mosaic-staining pattern will generally affect a small fraction of the cells present, and can generate false positive data if the tissue sample is heavily labeled. The methods of the present invention make it possible to avoid such false positives, by using appropriate tissue controls and/or cell controls rather than simply a xe2x80x9cnegative controlxe2x80x9d antibody.
In the fields of cancer research, diagnosis, and therapy, the morphologic evidence of cancer (or pre-cancer) together with the identification and measurement of specific cancer proteins in the same cells is a powerful combination. In principle, this combination of morphology and protein measurement permits one to know whether it is only the abnormal cells which are expressing specific proteins at particular moments and in known amounts in the natural history of the tumor being studied. Two things have been missing, however, from the set of tools needed to fully exploit this combination: (1) an accurate and reliable way to link individual foci of histologically and immunologically abnormal target cells (glandular crypt cells in the case of colorectal cancer); and (2) an accurate, objective and consistent quantitative method to score both the intensity/cell and the total immunopositive area (nuclear area in the case of p53). The present invention provides these missing elements (FIG. 1).
2. Role of p53 Protein in Cancer
The many roles of p53 in controlling the rate of cell proliferation and DNA repair at the G1/S phase of the cell cycle is widely appreciated. It acts to curb the effect of prior mutations that have occurred in pre-cancerous growths, such as adenomas. Less frequently acknowledged is p53""s role in maintaining the body""s xe2x80x9cback-upxe2x80x9d system of DNA-maintenance (diploidy) at both G1/S and the G2/M stages of the cycle [11, 12, 13].
Besides its importance in the rate of DNA replication, DNA repair/chromosome stability, and cell cycle arrest, p53 is one of the primary cellular reactants involved in the induction of programmed cell death: apoptosis. Normal p53 affects cell growth through its interaction, direct and indirect, with the cyclin-dependent kinase (cdk) regulatory pathways. It promotes apoptosis by stimulation of endonucleolytic enzyme attack upon chromosomes containing badly damaged DNA.[14] This occurs at the G1/S stage of the cell cycle. While p53""s cell growth arrest tumor suppressor function is temporary and reversible, the result of its apoptotic function is permanent. The sacrifice of the damaged/cancerous cells protects the whole organism against the cells"" undesirable continued replication and propagation of their heritable abnormalities.
3. Previous Methods for Quantitation of p53 Protein
There has been a great deal of effort expended to make detection and/or measurement of p53 levels simple and reliable. Numerous antibody-based histological reagents are now commercially available for immunohistochemical detection and estimation of p53 protein in tissue samples. A great deal of effort has also gone into attempting to correlate these measurements with tumor status and patient prognoses. The results of these efforts, to date, have been mixed.
At best, immunohistochemistry measurements on human tumor tissue are done in terms of arbitrary ordinal ranks or xe2x80x9cpercentage of intensely-staining cellsxe2x80x9d or the like. One review of the biochemical, immunological, and functional aspects of p53 reports that among mammalian cell cultures, transformed cell lines contain 10-100 times greater levels of p53 than non-transformed cells.[15] Studies indicate that this accumulation is due largely to protein accumulation, rather than increased gene dosage or RNA transcription levels. Using radio-labeling and a monoclonal antibody-bound affinity column, the p53 concentration in the lysates from 11 of 15 human tumor-derived or transformed cell lines was  greater than 5 times that of normal human cell lines.
Virally transformed cell lines exhibit extreme over-expression. For example, in SV40-transformed human fibroblasts p53 expression exceeded that of normal human cells by a factor of 2,250. In absolute terms, measured concentrations for these cell lines ranged from xe2x80x9cundetectablexe2x80x9d to 450 ng/mg. Normal cell cultures (human fibroblasts and human foreskin epithelial cells) had xe2x89xa60.2 ng p53/mg cellular protein.[16] Another study used two different types of fluorescent sandwich ELISA: a mutant-p53-specific (pAb 240), and a pantropic p53 monoclonal capture antibody (pAb 421) to measure p53 protein in 23 tumor cell lines, breast tissue extracts and 800 breast cancer patients sera. The mutant p53 for the extracts was all in the 0-2 ng p53/mg total protein range; and the sera were all negative. In two colorectal cancer cell lines having p53 mutations combined with deletion of the other allele and one colorectal cancer cell line having neither, the results [17] were as shown in Table 1 (ng p53/mg total cell lysate protein):
A similar study measured p53 levels in breast cancer tissue in immunostained paraffin sections and the cytosol extracts from the same tumors, It used the pantropic rabbit polyclonal antibody (CM-1) for the IHC and a sandwich-type ELISA incorporating the pantropic DO-1 monoclonal as the p53 solid-phase capture antibody and CM-1 as the detection antibody. There was a moderate correlation between the IHC and ELISA scores (Pearson R2=0.35, p less than 0.00001). However, IHC scoring [(ordinal rank for xe2x80x9cintensityxe2x80x9d)xc3x97(percentage p53+ cells)] is subjective and, therefore, impossible to compare with results from other studies, and it is difficult to use this information for prognosis when evaluating a given tissue sample. The IHC done upon the cancer cells"" nuclei was more sensitive than the ELISA, since it can distinguish not only cellular from stromal material, but also cancerous from non-cancerous tumor cells and p53+ from p53xe2x88x92 cancerous (or adenomatous) cells. The ELISA scores for the p53+ tissue sections were in ng p53/mg cytosol protein; the average value was 44 ng/mg protein with individual amounts ranging widely from 2-230 ng/mg.[18]
Another such study examined p53 levels in the soluble extracts of colon and gastric cancer tumors. The two-epitope, sandwich ELISA was employed using the DO-1 pantropic capture antibody for inactivated p53. The IHC was done on frozen fixed tissue with a panel of three different antibodies: one pantropic monoclonal (DO-1), one pantropic polyclonal (CM-1), and one mutant-specific (pAb 240). Again, the p53 range in the cytosol was similar: 0.1-2.3 ng p53/mg protein. Western blots done on the tumor tissue gave perfect+vs.xe2x88x92concordance between the DO-1 and pAb 240 antibodies in the Westerns; there was also 100% concordance by tumor among the assays (Westerns, pantropic ELISA, and IHC). In the same type of scoring as was done on the breast cancer tumors, the correlation was significant (Kendall""s r=0.75, p less than 0.002).[19]
Finally, the manufacturer of the mutant sandwich ELISA kit used herein for the calibration cell lines also reports detecting p53mut concentrations among 9 different mammalian cell lysates in the ng/mg range. Specifically, for the p53mut A431 cell line, also used herein, the reported result was 7 ng p53mut/mg.[20] One published study, however, using the same mutant ELISA, reported anomalous findings: 781 xcexcg p53mut/mg in the same A431 vulvar squamous carcinoma cell line, and very strong banding with a pantropic MAb Western blot, despite weak bands appearing in a Western blot using the mutant-specific PAb 240 antibody.[21] Clearly, there remain some difficulties with lab-to-lab variations in the execution of these assays.
In addition to measuring p53 quantities in mass and volume per tumor or cell lysate, it is possible to do so in terms of number of p53 molecules per cell, something that has been done very rarely. Measurement of the molecular concentrations of cancer proteinsxe2x80x94independently of their respective massxe2x80x94reveals the true ratios in which they combine in cellular reactions, providing insights into the stoichiometric chemistry of the cancer cell. It has been estimated that in normal cells the expression level is about 5,000 molecules p53/cell.[22] There is one other study of the number of p53 molecules/cell, which used flow cytometry to quantify the amount of p53 protein. These authors examined 10 different strains of bovine papilloma virus-transformed mouse fibroblasts and also one strain of non-transformed mouse fibroblast cells.[23] They found 2,947 molecules p53/cell in the non-transformed cells versus an average of 9,088 molecules p53/cell in the transformed fibroblasts. They also contrasted the levels of 10 cell lines within the transformed category (5 tumorigenic versus 5 non-tumorigenic), finding an average of 11,432 and 6,743, respectively (Mann-Whitney, p=0.0034).
There is a great deal more known about the levels of normal and inactivated p53 expression in human sera and plasma through the use of these same ELISA kits. There are examples of a statistically significant association between serum or plasma p53 ELISA and cancer/normal status.[24-26] There are instances of statistically significant correlations between the concentration of p53 in the blood and a patients diagnostic status for colorectal adenocarcinomas, adenomas, and normal controls, [27, 28] and multiple studies in which those levels decreased following surgical removal of the tumor (e.g., colon and breast, respectively). [29, 30]
There are examples of significant correlations between p53+ and p53xe2x88x92 status in tissue IHC, and xe2x80x9celevatedxe2x80x9d versus xe2x80x9cundetectablexe2x80x9d serum/plasma p53 in ELISA, or even continuous variable regression between the levels of p53 in tissue IHC and both mutant and pantropic ELISA analysis of serum.[31, 26] Examination of pancreatic adenocarcinoma paraffin sections stained with the DO-7 pantropic p53 monoclonal, combined with mutant p53 ELISA analysis of these same patients sera, has shown one of the strongest associations yet between blood and tissue p53 expression [32]. Of those people whose serum contained detectable mutant p53, 80% also had over-expressed p53 in their tumor tissue and had significantly greater blood concentrations Of p53mut, compared to the serum of those patients whose tissue was immuno-negative. Serum p53 in these cases was significantly greater than in healthy controls or patients with benign pancreatic conditions. Even though serum p53 concentrations did not correlate with those of more conventional markers such as CEA or CA19-9, it was significantly greater in those patients with existing metastases than in those without. Tissue staining appeared to be far more sensitive for p53 inactivation, than was the serum ELISA: 22% of the cancer patients were p53+ in the serum ELISA, while 46% of these patients were p53+ in the IHC. One study of banked lung cancer sera specimens, not only found significant agreement between DNA mutations, elevated p53+ IHC (DO-1), and p53mut serum ELISA (PAb 240) scores, but was able to predict future development of tumors based upon the detection of p53mut in the sera (positive predictive value=0.67, negative predictive value=0.83) [33]. There is some consistency among the cited studies regarding the ranges and averages of the concentration of inactivated p53 in human plasma or sera: typically, in the pg p53/mL range.
The invention provides a method for preparing cultured cells for immunostaining, which comprises the step of immobilizing said cells in a hydrophilic matrix that is non-liquid at room temperature (18-25xc2x0 C.). The matrix may be, for example, an aqueous gel of a polymer chosen from the group consisting of proteins, oligosaccharides, and poly(acrylamide), preferably gelatin, agarose, pectin, or poly(acrylamide). The matrix is more preferably an aqueous gel of xe2x80x9clow temperaturexe2x80x9d agarose. A typical low-melting point agarose, once dissolved in boiling phosphate buffered saline, remains in solution until cooled below 25xc2x0 C., and once solidified, only remelts above 65xc2x0 C. Such a matrix allows for convenient handling and avoids destroying any cell culture protein of interest from exposure to temperatures above normal physiologic range (37-40xc2x0 C.). Prior to immobilization, the cells may optionally be fixed by contacting them with a tissue fixative. Suitable tissue fixatives include formalin and Bouin""s.
The invention also provides a method of preparing calibration slides for a cell imaging densitometer. The method comprises the steps of immobilizing cultured cells in a hydrophilic matrix as described above, fixing the hardened matrix in a paraffin block in the usual manner, and sectioning the paraffin block into thin slices. The cultured cells are preferably preserved with formalin fixative prior to immobilization in the hydrophilic matrix. This method may be adapted to cryostat-sectioned frozen cultured cells and tissue, preserved with suitable fixatives e.g., acetone or ethanol, and embedded in a suitable tissue-freezing medium in place of paraffin. It may also be adapted to immunocytology specimens prepared as xe2x80x9csmearsxe2x80x9d from exfoliated patient cells or from clinical cell suspensions centrifuged at low speed, thus attached in either manner to microscope slides.
It was previously stated in PCT/US99/15743 that the embedded calibration cells must be fixed and treated in an identical fashion as the patients"" exfoliated cells or surgically-excised tissue. However, identical fixation was not in fact carried out in the examples described, and the superior results obtained with the present invention are due in part to the absence of such post-embedding fixation.
The invention also provides for visualizing a protein of interest on the calibration slide, wherein the slide is contacted with a first antibody. The first antibody may optionally be conjugated with a chromogenic or fluorogenic reagent. Alternatively, the slide may then be contacted with a second antibody, having binding affinity for the first antibody. The second antibody is also optionally conjugated to a chromogenic or fluorogenic reagent.
As an alternative to conjugation with chromogenic or fluorogenic reagents, the above antibodies may be conjugated to one of a pair of auxiliary affinity reagents. This permits binding, in a subsequent operation, of chromogenic or fluorogenic reagents which are conjugated to the other member of the pair. Suitable pairs of auxiliary affinity reagents include biotin-avidin and biotin-streptavidin. In this embodiment, the antibodies are preferably conjugated to avidin or streptavidin, which permits multimeric binding of biotin-conjugated chromogenic or fluorogenic reagents with a corresponding amplification in the signal. In an alternative embodiment, both the antibody and the chromogenic or fluorogenic reagent are conjugated to biotin, and they are contacted with one another in the presence of avidin or streptavidin.
A chromogenic reagent is a reagent that is itself highly colored, or that generates a colored dye or pigment upon exposure to specific chemicals or conditions. Examples of the latter include enzymes such as peroxidase.
A fluorogenic reagent is a reagent that generates light, upon exposure to specific chemicals or conditions, or that fluoresces upon exposure to light. Examples include enzymes which upon exposure to appropriate substrates generate luminescent or fluorescent products, such as peroxidase and luciferase, and fluorescent dyes such as fluorescein, brilliant red, rhodamine, and the like. Numerous such reagents and dyes are well-known in the art, and are anticipated to be useful in practicing this invention.
The particular embodiment described herein employs biotin and avidin as the auxiliary affinity reagents, peroxidase as the chromogenic reagent, hydrogen peroxide and 3,3xe2x80x2diaminobenzidine (DAB) as substrates [see U.S. Pat. No. 4,684,609], and image densitometry as the measurement method. It will be readily understood by those skilled in the art that fluorometric (e.g. photon-counting) methods with a fluorescence microscope (e.g. a CCD camera-equipped microscope) will be equally operative if the conjugated reagent generates rather than absorbs light. The chromogenic or fluorogenic reagent (or reaction product) will preferably absorb or emit light within a narrow enough wavelength range that a second chromogenic or fluorogenic reagent or product, emitting or absorbing in another wavelength range, will be usable without interference. Many such reagents are known in the art, and most are anticipated to be useful in practicing the present invention.
The invention further provides a method for measuring the amount of a protein of interest in a cell or a cell organelle. The method comprises the steps of affixing the cell to a microscope slide, optionally in the form of a paraffin block section, staining the cell with an immunohistochemical stain such as the conjugated antibodies described above, and measuring with a cell imaging densitometer (or fluorimeter) the area and density of the stain within the cell or cell organelle. The amount of stain within the cell or cell organelle is proportional to the summed optical density, which is most conveniently calculated with the software associated with the instrument. The summed optical density is then converted into the amount of protein of interest by reference to a calibration slide prepared as described above and stained concurrently with the same immunohistochemical stain. The amount of protein of interest actually in the cells on the calibration slide is measured by an independent assay of said protein in a sample of the calibration cells. The independent assay method may be any assay that is quantitative and specific for the protein of interest, such as an ELISA or Western blotting assay, preferably an ELISA. Provided that the molecular weight of the protein of interest and its molecular concentration in the calibration cells has been determined, measurement of the average volume of the diseased cells expressing the protein in the body allows for the estimation of the number of such molecules per cell. The phrase xe2x80x9camount of protein of interestxe2x80x9d is intended to encompass amounts measured in mass units, moles, or numbers of molecules; this amount may be expressed relative to any convenient measure, such as per cell, per cell organelle, per patient, per tumor, or per unit volume of tissue or body fluid.
The protein of interest may be a tumor-associated protein, and the cell may be a tumor cell, as in the examples below. Other proteins of interest, which may be associated with disease states, which may be expressed in recombinant, gene-activated, or endogenous cells for a therapeutic purpose, or which may be of research interest, may be quantitated as well, provided only that appropriate cell lines and specific antibodies are available to the practitioner, or can be prepared.
The invention also provides a method of calculating a patient""s body burden of a protein of interest. This method comprises the steps of measuring the amount of the protein of interest in one or more cells taken from one or more of the patient""s tumors, by the method described above, and converting the amount of protein so determined into the amount of protein in the tumor itself. This is readily done by estimation of tissue or tumor volume from measured tissue or tumor dimensions. By adding together the amount of protein in each tissue or tumor, the total amount of protein in all the patient""s tissue or tumors is obtained, and this is the patient""s body burden of the protein.
The invention also provides a simple and inexpensive method for measuring the area of a feature of interest that is visible in the field of view of a microscope equipped with a video camera, where software providing this function is either not available or unsuitable. A field finder (a microscopic printed grid) is placed over the microscope slide, which, with appropriate magnification, results in the appearance of a grid with easily visible squares on the monitor. An outline of the stained region of interest, which is imaged on the monitor, is traced on a material having a printed grid on its surface, such as a sheet of graph paper. The outline is then cut out and the cutout piece of material weighed. With knowledge of the mass of the material per unit area, the mass of the cutout may optionally be converted to area of material. By outlining, cutting, and weighing a rectilinear piece of the same material corresponding to a known number of grid squares on the monitor, the area (or mass) of the cutout may be converted into actual area on the slide, as measured by the field finder. The xe2x80x9cconversion factorxe2x80x9d is the mass of the material per unit area of the field finder as imaged on the monitor. Any graph paper or similar material may be used, provided that the density of the material is sufficiently uniform to provide a reliable correlation between mass and surface area. The microscope objective and dimensions of the matrix visualized on the monitor are usually specified by the user using a menu from the CID software provided. FIG. 2 is an overview of sequence of the immunohistologic measurements and how one measurement is derived from another. The (mm2) area measurement procedure co-listed in step one in FIG. 2 is described in more detail below [Cell Imaging Densitometry Measurements, including Table 7].
The invention also provides for a method of predicting the clinical outcome of cancer for a patient, which involves providing a statistically derived continuous function that relates the amount of a tumor-associated protein within the patient""s tumors, as measured by the method of this invention, to clinical outcome in a population of patients with the same cancer. By measuring the amount of the tumor-associated protein in the patient""s tumor cells by the same method, and by referring to the continuous function provided, clinical outcome may be predicted with improved reliability over prior art methods.
For cancer prognosis, especially for adenocarcinoma, the tumor-associated protein is preferably a mutant p53 (p53mut). In those cancers which are partly due to a loss of p53 function, the expected increase in multi-drug resistance and reduction in the effectiveness of anti-angiogenesis drugs would make such patients poor candidates for these treatment options. Alternatively, the replacement of the presumably defective p53 gene with a functional gene coding for p53, by means of genetic therapy, is a promising approach.[34-36] Administration of genetic therapy with p53-encoding DNA, which is based upon restoration of p53 expression in tumor cells, presupposes that the prospective patient""s tumors are expressing p53mut. It would, therefore, be useful to know beforehand the p53 status of the patient, both for selecting a patient population for clinical trials and for guiding administration of genetic therapy to patients among the public. Other p53-specific therapies are under investigation, including antisense DNA therapies and anti-p53 antibodies, which are directed toward reducing the tumorigenic effects of p53mut. The present invention provides a method of selecting patients for p53-specific therapies, and p53 genetic therapy with p53-encoding DNA, based upon a quantitative measure of p53mut concentrations in the patient""s tumor cells and/or the patient""s p53mut body burden. The present invention provides, as well, a method of monitoring the effectiveness and progress of such therapies, again by quantitative measure of p53mut concentrations in the patient""s tumor cells, total p53mut body burden per patient, or its derivative surrogate measure: the concentration of p53mut in his or her blood.
The present invention employs cultured cells, preferably inexpensive standard human tissue cell lines standardized according to quality controls performed by the American Type Culture Collection. The calibration cells express a protein of interest at a reproducible level that can be easily and accurately measured. In the first instance, these cells can be used, simply, as xe2x80x9cpositivexe2x80x9d and xe2x80x9cnegativexe2x80x9d immunostaining xe2x80x9cbatch controlsxe2x80x9d. In this aspect the changing intensity of their staining improves CID by helping the operator decide which cells within the tissue sample to score. In this preliminary stage of protein quantitation they have not yet been assigned any measured absolute biological values, merely their average OD/cell. In this aspect the invention reduces xe2x80x9cmisclassification errorxe2x80x9d (categorical false positives and negatives) in immunohistopathological analyses. It simply helps in answering the question: xe2x80x9cDoes this tissue section contain any of the abnormal protein to be analyzedxe2x80x9d?
The second aspect of the invention is to utilize these same control cells as xe2x80x9ccalibration cellsxe2x80x9d in order to translate the optical density units of cell imaging into biologically meaningful measures of protein dosage at the level of the individual organelle, cell, tumor, tissue, or patient. The method provides, for the first time, batch-specific standard curves expressed as a continuous quantitative function that are applicable to any lab or immunostaining procedure. These continuous functions are superior to the ordinal ranking methods of the prior art, whichxe2x80x94either with or without CIDxe2x80x94ultimately forced one to assign ad hoc relative categories to degrees of IHC staining. The quantitative scoring method of the present invention provides more objectivity, accuracy, reproducibility, biological validity, and consistency among observers than has hitherto been possible. By creating absolute xe2x80x9cinterval-levelxe2x80x9d units of measurement, the present invention makes it possible to apply the discriminatory power and precision of multivariate parametric statistical tests in cellular protein quantitation.
The embodiment of the invention in the examples below relates to p53 in a particular xe2x80x9ctestxe2x80x9d population with colorectal dysplasia; the analysis of their tissue and blood demonstrates the potential of the present invention to improve protein quantitation, cancer diagnosis specificity, tailor and monitor oncology treatment, and to provide a tool for more exact and powerful prognosis. However, the invention can be applied to any disease-associated protein for which for adequate cultured cell controls and suitable antibodies exist.
The present invention makes use of a method of cultured cell preparation, which immobilizes the cells in a solid hydrophilic matrix. The resulting matrix of immobilized, cultured cells behaves much like a sample of tissue, and can be fixed, sectioned, and stained in the same manner as a tissue sample. The hydrophilic matrix may be based on protein, e.g. gelatin, or on a hydrophilic polymer such as acrylamide or an acrylamide derivative, but is preferably a low-melting solution of an oligosaccharide such as pectin or agarose. More preferably the matrix is agarose, and most preferably a low-temperature agarose gelatin. Immobilization of cells in an inert, stable, physiologic-temperature matrix avoids the incomparability of frozen cell pellets compared to formalin-fixed, paraffin-embedded tissue with respect to protein denaturing, disruption of cell morphology, differing antibody affinities, and fixation artifacts. The use of thermally meltable matrix materials is preferred over the use of chemically polymerized polymers, such as acrylamide, because of the simple and reliable process of solidification provided by temperature control.
Although the matrix of immobilized cells may be treated in the same manner as a tissue sample, it is preferable that it not be subjected to fixation beyond the minimum necessary to preserve structural features and prevent diffusion of the protein of interest away from those structures. Preferably, the fixation will be in normal buffered formalin (NBF), or the equivalent treatment, and most preferably this will be carried out on the cultured cells prior to immobilization in the hydrophilic matrix. The fixation time is preferably less than four hours in NBF, more preferably less than two hours, and even more preferably less than one hour in NBF (or the equivalent treatment). Most preferably the fixation is conducted in NBF for less than thirty minutes, for example ten minutes or less. Treatments equivalent to fixation with NBF, such as for example fixation with acrolein, glutaraldehyde, or cyanuric chloride, are known to those of skill in the art.[125, 130]
In a preferred embodiment, fixation and staining of the cultured cells are carried out with the same reagents, but are optimized separately from the fixation and staining of the tissue specimen upon which protein quantitation is being carried out. This is in contrast to prior art methods which emphasized the importance of identical treatment at all stages of processing.
Storage of colorectal adenocarcinoma tissue blocks for 13 years reportedly has no appreciable effect on the levels of nuclear accumulation of both p53 and BCL-2 proteins as scored by cell imaging densitometry.[37] This was true for the proportion of positive (xe2x89xa710% cell nuclei) cases, as well as the average intensity/cell, even though the more sensitive and reliable xe2x80x9cantigen retrievalxe2x80x9d treatment was not used for the p53 staining. After fixing and paraffin embedding, the immobilized cells of the present invention provide a reference sample that can be reasonably expected to be as durable and permanent as any paraffin-embedded tissue sample. A single cell pellet from a typical 75mm2 culture flask can provide hundreds of calibration paraffin sections, which are suitable for commercial production and sale.
By way of illustration, the application of the invention to the quantitation of mutant p53 protein in colorectal adenomas and adenocarcinomas, and the resulting improvement in accuracy of diagnosis and prognosis, is described below.
Thus, one object of the invention is to provide a method for preparing cultured cells for immunostaining, which comprises the step of immobilizing the cells in a hydrophilic matrix. Preferably, the matrix is an aqueous gel of a polymer chosen from the group consisting of proteins, oligosaccharides, and poly(acrylamide). In specific embodiments, the matrix is an aqueous gel of a polymer chosen from the group consisting of gelatin, agarose, pectin, and poly(acrylamide). Preferably the matrix is an aqueous gel of agarose, and the agarose is most preferably a low-melting point agarose.
It is another object of the invention to provide a method of preparing calibration slides for a cell imaging densitometer, comprising the steps of:
(a) immobilizing cultured cells in a hydrophilic matrix;
(b) placing the matrix in molten paraffin;
(c) cooling the molten paraffin until it solidifies; and
(d) without substantial intervening fixation, sectioning the solidified paraffin containing the immobilized cells into at least one thin slice suitable for optical microscopy.
In this method, the cultured cells are preferably contacted with a tissue fixative prior to immobilization in the hydrophilic matrix. The method preferably further comprises the step of contacting the paraffin slice with a first antibody. The first antibody is preferably conjugated to a chromogenic or fluorogenic reagent.
In an alternative embodiment, the method further comprises the step of contacting the paraffin slice with a second antibody having binding affinity for the first antibody, the second antibody preferably being conjugated to a chromogenic or fluorogenic reagent.
In yet another embodiment, the first antibody is conjugated to biotin. Preferably, the slice is then contacted with a biotinylated chromogenic or fluorogenic reagent in the presence of avidin or streptavidin.
It is another object of the invention to provide a method for measuring the amount of a protein of interest in a cell or a cell organelle, comprising the steps of:
(a) affixing said cell to a microscope slide;
(b) staining said cell with an immunohistochemical stain;
(c) measuring with a cell imaging densitometer the area and density of the stain within the cell or cell organelle;
(d) calculating the summed optical density of the stain within the cell or cell organelle; and
(e) converting the summed optical density into the amount of protein of interest, by reference to
(i) a calibration slide prepared according to the method of the invention described above, and stained with the same immunohistochemical stain as was used in step (b); and
(ii) the amount of protein of interest actually in the cells or organelles on the calibration slide, as measured by an assay of the protein of interest in a sample of the cells.
In the above-described methods, the protein of interest is preferably a tumor-associated protein, and preferably the cell is a tumor cell. The tumor cell is preferably fixed in a paraffin tissue section.
It is yet another object of the invention to provide a method of calculating a patient""s body burden of a tumor-associated protein of interest, comprising the steps of:
(a) measuring the amount of the protein of interest in one or more cells taken from one or more of said patient""s tumors, by any of the methods described above;
(b) converting the amount of protein determined in step (a) into the amount of protein in the tumor from which the cell was obtained; and
(c) adding the amount of protein in each tumor to obtain the total amount of protein in the patient""s tumors.
Another object of the invention is to provide a method of calculating the probable clinical outcome of cancer for a patient, comprising the steps of:
(a) providing a statistically-derived continuous function relating the body burden of a tumor-associated protein, or the amount of a tumor-associated protein within the patient""s tumor cells, to clinical outcome, in a population of patients with the same cancer;
(b) measuring the patient""s body burden of the tumor-associated protein, or the amount of a tumor-associated protein within the patient""s tumor cells, by any of the methods described above; and
(c) using the continuous function provided in step (a) to calculate the probable clinical outcome.
In all of the above methods involving a tumor-associated protein, a preferred tumor-associated protein is p53mut.
Another object of the invention is to provide a method of selecting a patient for p53-specific therapy, which comprises measuring the patient""s body burden of the tumor-associated protein, or the amount of a tumor-associated protein within the patient""s tumor cells, by any of the methods described above. A preferred p53-selective therapy is genetic therapy with p53-encoding DNA.
Another object of the invention is to provide a method of monitoring the effectiveness or progress of a p53-specific therapy, which comprises measuring the patient""s body burden of the tumor-associated protein, or the amount of a tumor-associated protein within the patient""s tumor cells, on a per-cell basis, by any of the methods described above. Preferred p53 selective therapies include in situ treatment with p53 peptides or p53wt DNA.